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Research Service, Albuquerque Veterans Affairs Medical Center, Albuquerque, NM, 87108; Departments of
Molecular Genetics and Microbiology and
Pathology, University of New Mexico, Albuquerque, NM 87131;
§
Department of Microbiology, Dartmouth Medical School, Hanover, NH 03755; and
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Departments of Infectious Diseases and Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The possibility that phagocytes, particularly PMN, could facilitate S. aureus infection has been raised by other investigators (11, 12, 13, 14, 15). In vitro studies from the 1950s and 1960s demonstrated that pathogenic strains of S. aureus could survive for long periods of time inside both PMN and monocytes isolated from different animals and humans (12, 13, 14). These studies led investigators at the time to speculate that both intracellular survival and extracellular multiplication play important roles in the pathogenesis of S. aureus infections (11). In this regard, recent experiments assessing invasion and intracellular survival of S. aureus in endothelial cells (16), epithelial cells (17), and osteoblasts (18) have suggested that intracellular survival could contribute to the persistence of the pathogen in S. aureus-induced endocarditis, bovine mastitis, and osteomyelitis. Although these in vitro studies speculate on the adaptive advantage of intracellular survival in nonprofessional phagocytes, no studies have assessed whether survival of S. aureus inside PMN occurs in vivo and whether this can promote infection.
Therefore, the purpose of this work is to test the hypothesis that survival of S. aureus inside PMN contributes to the pathogenesis of this infection. As with earlier in vitro studies (11, 12, 13, 14, 15), we found that PMN isolated from the site of infection contained viable intracellular bacteria. Moreover, we show that the ability of S. aureus to exploit the host’s inflammatory response is regulated by sar, a global regulator of multiple virulence factors in this organism (2). These data demonstrate that although necessary for infection control, appropriately stimulated PMN can create an environment that promotes the intracellular survival of this pathogen to the detriment of the host.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6J and BALB/cByJ mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice deficient in integrin-associate protein (IAP, CD47) were backcrossed onto either C57BL/6J or BALB/cJ strains as described (19). All studies were approved by the Institutional Animal Use Committees. For infection studies, mice were age and gender matched. To obtain bone marrow leukocytes, femurs were removed aseptically and flushed with HBSS/0.2% human serum albumin (HSA). PMN were isolated from bone marrow leukocytes using NIM2 isolation media (Cardinal Associates, Santa Fe, NM) following the manufacturer’s instructions and resuspended in endotoxin-free sterile saline (McGaw, Irvine, CA). The cells were 75–80% PMN as determined by staining with 0.1% cresyl violet.
Bacterial strains
S. aureus strain DB, a microencapsulated clinical blood isolate, and a sar- transposon-induced mutant of DB, 11D2, were characterized as described (2, 3, 7). 11D2 is significantly less virulent than DB in a rabbit model of S. aureus-induced endocarditis (3). RN6390, a prototypic, type 5 microencapsulated strain, was characterized as described (7). Strain SA1, an encapsulated and TSST-1-positive clinical isolate, was provided by Dr. J. Lee (Harvard University, Cambridge, MA) (20). Type III Streptococcus pneumoniae was purchased from the American Type Culture Collection (ATCC) (no. 10813). Bacteria were cultured on either blood agar or Columbia salt agar.
Lethal and sublethal infection models
S. aureus infection of mice was performed as
described previously (9). Bacteria were adjusted to the
desired inoculum spectrophotometrically before injection
(OD620 = 1.6 for 
1.0 x
109 CFU/ml), and the CFU were confirmed by serial
dilution and culture on blood agar (Becton Dickinson, Cockeysville,
MD). Survival in response to i.p. challenge and the number of WBC per
ml, percentage of PMN, number of PMN per ml, and the
log10 CFU/ml of a 5-ml peritoneal lavage (HBSS
with 0.2% HSA and 5 mM EDTA) obtained 24 h after i.p. challenge
(both lethal and sublethal CFUs) were performed as described
(9). The challenge CFUs are given in the figure legends.
Mice were sacrificed when moribund as assessed by decreased body
temperature, impaired ambulation, the inability to remain upright,
prolonged lethargy, and prolonged inappetance. At the lethal dose, the
moribund condition was reached by 24–72 h.
Gentamicin protection assay of S. aureus associated with PMN in vivo
To determine whether viable S. aureus organisms
associated with PMN were intracellular, we performed a gentamicin
protection assay on the extracellular and PMN-associated bacteria in
the peritoneal lavage obtained from mice 24 h after i.p.
challenge. Gentamicin kills S. aureus but is limited in its
ability to gain access to intracellular organisms. The peritoneal
lavages from several infected mice were combined, and the PMN were
purified by depleting lymphocytes and macrophages by negative selection
as described (10). The Abs used for depletion were 30H12
(rat IgG2b anti-mouse Thy1.2), GK1.5 (rat IgG2b anti-mouse
CD4), and F4/80 (rat IgG2b anti-mouse macrophage). Ab-coated cells
were incubated with 0.4 ml goat-anti-rat IgG magnetic particles
(PerSeptive Diagnostics, Cambridge, MA) in cold HBSS buffer for 20 min
on ice. The percentage of PMN in the peritoneal exudate was increased
from 82% to 98% depending on the experiment, and PMN viability
was >95%. Aliquots were taken from both the extracellular supernatant
and the PMN suspension to assess log10 CFU before
incubation with 100 µg/ml gentamicin at 37°C. After 2 h, the
PMN and the extracellular bacteria were washed twice to remove the
gentamicin and resuspended to the original volume of the lavage;
aliquots were taken to assess the log10 CFU. To
ensure that PMN-associated bacteria were capable of being killed by an
antibiotic, the cell pellets were incubated with 1 mg/ml rifampin. This
treatment significantly reduced the PMN-associated CFU.
Use of PMN containing viable S. aureus to establish infection
To ascertain whether PMN containing viable S. aureus
can contribute to infection, PMN were purified from peritoneal lavages
24 h after infection with 1 x 106 SA1,
treated with 100 µg/ml gentamicin for 2 h, and washed twice; an
aliquot was taken to determine the CFU and was injected i.p. into
wild-type mice. The number of PMN injected was determined to give
1 x 106 CFU of SA1. As a control, the
equivalent number of uninfected PMN purified from the bone marrow were
also injected i.p. After 24 h, a lavage was performed and the
total number of PMN and the total log10 CFU were
assessed.
Electron microscopy
To assess the intracellular locale of bacteria taken up by PMN, peritoneal exudate cells obtained 24 h after i.p. challenge with DB, the sar- mutant 11D2, or SA1 were analyzed by transmission electron microscopy. The cells were fixed as a pellet in 2% glutaraldehyde, osmicated in 2% osmium tetroxide, stained en bloc with 2% uranyl acetate, treated with propylene oxide, and Epon embedded. Ultrathin sections were placed on copper grids, stained with uranyl acetate and lead citrate, and viewed in an Hitachi 600 transmission electron microscope (21). Photographs of representative fields (two experiments for each strain) were analyzed, and the percentage of bacteria in large, spacious vacuoles, in tight phagosomes, and free in the cytoplasm (non-membrane bound) was quantified by two independent observers.
mAb treatment and chemokine administration
To partially or fully deplete PMN, we used administration of rat IgG2b anti-Gr-1 mAb RB6-8C5 as described by others (8, 22). The RB6-8C5 hybridoma was obtained from DNAX (Palo Alto, CA) and an isotype control (SFR3) which does not recognize murine cells was purchased from ATCC. The Abs were purified from culture supernatant by ammonium sulfate precipitation and protein G-Sepharose (mAb Trap GII; Pharmacia Biotech, Alameda, CA). Before injection, the purified Abs were subjected to buffer exchange against sterile endotoxin-free normal saline to ensure the absence of endotoxin as previously described (9). Mice were injected i.v. via the tail vein with 5–250 µg of mAb RB6 vs control SFR3 to assess the effect on PMN number and on the host response to infection. For partial depletion, 5–10 µg mAb RB6 per mouse consistently depleted 25–33% of bone marrow PMN. At this concentration, SFR3 had no effect. For full depletion, 100 µg or greater were required to deplete >90% of PMN for 72 h.
To promote PMN migration into the site of infection, we administered
the murine CXC chemokines, macrophage-inflammatory protein (MIP)-2 and
KC (R&D Systems, Minneapolis, MN). As a control we administered the
murine CC chemokine MIP-1a. Recombinant MIP-2, KC, and MIP-1
were
reconstituted in buffer containing 0.2% HSA and were stored at
-70°C in sterile tubes precoated with 0.2% BSA-PBS. The stock was
diluted with endotoxin-free sterile normal saline to deliver 400 ng in
100 µl vs a vehicle control of buffer diluted into saline. To assess
CXC chemokine levels in lavage samples by ELISA (R&D), the samples were
centrifuged, sterile-filtered, and stored at -70°C in tubes
precoated with 0.2% BSA.
In vitro uptake of S. aureus by human PMN
To ascertain whether S. aureus can survive
intracellularly in human PMN taken up in vitro, we incubated human PMN
(1 x 107), isolated as described
(23), with 1 x 108 bacteria
(strains DB, 11D2, or SA1) in 1 ml of buffer (HBSS containing 0.5 mM
Ca2+, 2.5 mM Mg2+, and 1%
sterile, endotoxin-free HSA) in the presence or absence of various
concentrations of recombinant human IL-8 or control chemokines (R&D
Systems). The optimal concentration of IL-8 for uptake was 500 ng/ml.
Minimal uptake of the bacteria was observed in the absence of IL-8.
Controls included 500 ng/ml of the CC chemokine, MIP-1, and 15 ng/ml
phorbol dibutyrate. To control for the role of blood-derived opsonins,
the effect of 10% autologous fresh human serum from a donor with
agglutinating Ab against strain DB was also evaluated. After 4 h
at 37°C, the extracellular and PMN-associated bacteria were subjected
to gentamicin protection as described above. At this time, PMN
viability was >95%.
Statistical analysis
Data are presented as the means ± SEM. Statistical analyses were determined by Fisher’s exact test for the survival studies and by the Mann-Whitney U test for nonparametrics for all other analyses, using Statview for MacIntosh.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess whether viable S. aureus could be detected
inside PMN isolated from a site of infection, we subjected both the PMN
and the extracellular supernatant of peritoneal lavages obtained
24 h after i.p. challenge of wild-type mice to gentamicin
treatment. S. aureus is susceptible to gentamicin, but
because gentamicin does not readily cross plasma membranes,
intracellular organisms are protected from optimal killing. For strain
DB, a microencapsulated clinical blood isolate (2, 3),
equivalent numbers of bacteria were extracellular and PMN-associated
(Fig. 1
A). However, the
extracellular DB were readily killed by gentamicin whereas the
PMN-associated organisms were not significantly affected, indicating
that they remained viable inside the PMN (Fig. 1A).
Equivalent data were obtained with an additional microencapsulated
strain RN6390 as with DB (data not shown). If intracellular survival
represents a virulence mechanism for S. aureus, then mutants
with attenuated virulence should be less able to survive inside PMN. To
address this, we examined a transposon-induced mutant of DB, 11D2,
which has attenuated virulence in several animal models due to the loss
of a regulatory locus, sar, which governs the synthesis and
secretion of several virulence factors, including matrix protein
binding adhesins (2, 7). The sar- mutant had
significantly fewer organisms associated with PMN, and these were not
protected from gentamicin killing (Fig. 1B). To assess
whether this was a property of other Gram-positive bacteria, we
challenged mice with 1 x 103 CFU type III
Streptococcus pneumoniae and assessed whether PMN isolated
from the site of infection contained viable bacteria. No
gentamicin-resistant organisms were detected associated with PMN (data
not shown). These data indicate that at 24 h after the initiation
of infection, wild-type S. aureus, but no other
Gram-positive bacteria, remain viable inside PMN and that their
intracellular survival is regulated, at least in part, by the
sar regulatory locus.
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107 CFU were PMN associated and
resistant to gentamicin killing equivalent to what we observed with DB
(Fig. 1
C). These data indicate that capsule type does not
affect the ability of S. aureus to gain access to an
environment inside PMN that permits its survival.
The above data indicated that S. aureus remain viable inside
PMN; however, this viability could be transient. To assess whether
these infected PMN are capable of transferring infection, we injected
SA1-infected, gentamicin-treated PMN i.p. into naive mice. As shown in
Fig. 1D, injection of PMN containing
106 viable SA1 was sufficient to cause an
additional influx of PMN (p = 0.0044) and
result in significant growth of the pathogen (p
= 0.0082) 24 h after administration of the cells. Injection of an
equivalent number of uninfected PMN purified from the bone marrow
(4.55 x 106 injected PMN) resulted in a
reduction in the number of PMN present at 24 h (0.79 ±
0.09 x 106 recovered PMN, n
= 3), indicating that the presence of uninfected PMN is not sufficient
to recruit additional PMN. The bacterial burden in the lavage consisted
of both extracellular and intracellular organisms (data not shown).
These data demonstrate that intracellular S. aureus survive
long enough inside PMN to transfer infection.
sar+ and sar- S. aureus are in physically different intracellular vacuoles
To examine the location of S. aureus inside PMN
isolated from a site of infection, we performed transmission electron
microscopy on PMN from peritoneal lavages taken 24 h after i.p.
challenge with sar+ strains DB and SA1 and sar-
11D2. Intracellular bacteria were found in large vacuoles, "spacious
phagosomes," (Fig. 2 A and
B), free in the cytoplasm (Fig. 2C), or in small
vacuoles, "tight phagosomes" (Fig. 2D). For both DB
(Fig. 2A) and SA1 (Fig. 2B), the majority of the
bacteria were in large vacuoles similar to spacious phagosomes created
by macropinocytic uptake (24, 25, 26) (76.9 and 69.6%,
respectively). A minority were in tight phagosomes (12.8% for DB and
21.7% for SA1) or free in the cytoplasm (10.3% for DB and 8.7% for
SA1). Individual PMN often contained bacteria in all three
intracellular locales (data not shown). In contrast, sar-
11D2 were primarily in tight phagosomes (97.2%) and rarely in large
vacuoles (2.8%) or free in the cytoplasm (2.8%). Some of the
wild-type bacteria were in membrane-bound vacuoles (Fig. 2B,
black arrows) whereas others were in vacuoles where the membrane had
either totally or partially degraded (Fig. 2, A and
B, white arrows). This same phenomenon was recently observed
with S. aureus taken up by epithelial cells in vitro and was
thought to precede escape of the bacteria into the cytoplasm
(17). These data indicate that sar+ strains,
which are able to survive inside PMN, are primarily located in
macropinosome-type organelles (spacious phagosomes) and that
sar- strains, which do not survive inside PMN, are in small
vacuoles with tightly apposed membranes (tight phagosomes). Therefore,
sar regulates intracellular survival of S. aureus
by influencing the intracellular locale of the internalized
bacteria.
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If survival of S. aureus inside PMN represents a
significant contribution to infection with this pathogen, then limiting
access of the organism to this intracellular site should be beneficial.
To test this hypothesis, we assessed survival in response to S.
aureus-induced peritonitis and sepsis in IAP-deficient mice
(19). IAP is a multiply membrane-spanning member of the Ig
gene family (27), which modulates PMN migration across
endothelial cells (28). We have published previously that
IAP deficiency limits PMN migration in response to E.
coli-induced peritonitis resulting in an increased bacterial
burden and increased mortality of the mice (19).
Therefore, if reducing the number of PMN in S.
aureus-induced peritonitis and sepsis would be beneficial, then
IAP-deficient mice should have enhanced host defense against S.
aureus. To examine this, we injected IAP+/+
wild-type and IAP-/- C57BL/6 mice with a lethal
dose of SA1 (1–2 x 106 CFU) into the
peritoneum and assessed survival. In contrast to what we observed with
Escherichia coli (19),
IAP-/- mice had significantly enhanced survival
in response to SA1-induced sepsis (Fig. 3A, p =
0.0055). To assess the host response to infection at a time when most
wild-type mice become moribund, we measured the leukocyte number,
including the number of PMN, and the bacterial burden at the site of
infection 24 h after infection. IAP-/-
mice had significantly fewer PMN (p = 0.0045)
and an 1.5 log reduction (p = 0.0044) in CFU
at the site of infection. The appearance of the
IAP-/- mice also correlated with the increased
survival and reduced bacterial burden; they were grooming themselves,
eating, drinking, and were less lethargic than
IAP+/+ mice. These data indicate that reduced PMN
migration into the site of S. aureus infection due to IAP
deficiency results in enhanced survival of the host and a reduced
bacterial burden.
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Because IAP is expressed on all cells (27), the enhanced survival of the IAP-/- mice could be related to other consequences of IAP deficiency. To address this, we limited PMN influx by partially depleting PMN by injection of mAb anti-Gr-1 RB6-8C5 (22). The essential role of PMN in several murine models of infection, including S. aureus-induced arthritis (8, 22), has been demonstrated by total ablation of PMN with this Ab. In preliminary experiments, we found that i.v. administration of 5–10 µg of purified mAb RB6 vs an isotype control, SFR3, depleted 25–33% of bone marrow PMN, which resulted in a 50–66% reduction in the number of PMN migrating into the peritoneum in response to infection. A concentration of 100 µg depleted 90–100% of PMN migrating into the peritoneum. These single doses were sufficient to alter the number of PMN for 72 h.
To confirm the results of others that PMN play an essential protective role in S. aureus infection (8), we assessed the effect of complete ablation of PMN on S. aureus-induced peritonitis/sepsis. Wild-type mice were treated with 100 µg mAb RB6 to fully deplete the PMN migrating into the peritoneum 24 h before a sublethal dose of SA1. Greater than 90% depletion of the PMN resulted in a significant increase in the number of bacteria as compared with an Ab control (7.58 ± 0.05 log CFU vs 4.99 ± 0.15 log CFU, p = 0.002, n = 4) (data not shown). The RB6-treated mice were moribund in appearance, confirming that total PMN ablation is able to convert a sublethal infection to a lethal infection. These data indicate that PMN do play an essential protective role in S. aureus infection.
To test the effect of partial PMN depletion on survival against a
lethal dose of S. aureus, we treated wild-type mice with 10
µg mAb RB6 or isotype control 24 h before i.p. challenge with
2 x 106 CFU of SA1. By 48 h, all of
the control mice were moribund and succumbed to the infection. In
contrast, the mAb RB6-treated mice all survived and appeared normal
after 2–3 days (Fig. 4A,
p = 0.004). To confirm that the enhanced survival of
the mAb RB6-treated mice was due to a reduced bacterial burden and that
this concentration of Ab only partially depleted PMN in the peritoneum,
we assessed the number of PMN per ml and the CFU per ml in a lavage
from the site of infection. Both of these parameters were significantly
reduced in the mAb RB6-treated mice (Fig. 4B, hatched bars).
Therefore, enhanced survival of the mAb RB6-treated mice coincided with
an 66% decrease in PMN number and with a 2000-fold reduction in
the number of bacteria at the site of infection. To confirm that this
benefit could be observed with other S. aureus strains and
that it was dose responsive, we assessed the effect of two doses of RB6
on peritonitis induced by the microencapsulated strain DB. As shown in
Fig. 4D, there was a dose-dependent effect of RB6 on
DB-induced mortality (p = 0.03). In addition,
we assessed whether RB6 treatment affected gentamicin-resistant,
PMN-associated CFU. The RB6-treated mice had significantly fewer
gentamicin-resistant, PMN-associated DB present at the site of
infection than did the mice treated with the control SFR3 (2.84 ±
0.4 vs 5.89 ± 0.2 log CFU, n = 4). These data
demonstrate that partial depletion of PMN significantly enhances host
defense against S. aureus-induced peritonitis and sepsis and
that this benefit arises at least in part by reducing the number of
viable organisms inside PMN.
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C,
p = 0.004). These data indicate that the protection
conferred by partial PMN depletion can be reversed by administration of
purified PMN from either wild-type or IAP-/-
mice. The ability of IAP-/- PMN to reverse this
protection is not unexpected because IAP-/- PMN
are not completely devoid of the ability to migrate into the peritoneum
(Fig. 3B (19)), and their migration is greatly
facilitated in IAP+/+ mice (H. Gresham and F.
Lindberg, unpublished observations). To confirm that the decreased
survival with the infusion of PMN resulted in increased PMN migration
into the peritoneum and an increased bacterial burden, we performed a
lavage 24 h after SA1 challenge of RB6-treated mice in the
presence and absence of additional wild-type or
IAP-/- PMN. The number of both types of PMN was
significantly increased as was the bacterial burden and both were
similar to the values in SFR3-treated mice depicted in Fig. 4B (data not shown). Taken together, these data indicate
that the protection provided by low dose mAb RB6 and IAP deficiency
against S. aureus-induced peritonitis and sepsis is
specifically mediated by their effects on PMN. A trivial explanation for these data could be that IAP deficiency or mAb RB6 stimulates bactericidal killing by murine PMN, thus enhancing host defense. To address this, we assessed killing of DB by IAP+/+, IAP-/-, and RB6-treated wild-type PMN in vitro. Incubation of 2 x 106 DB with 1 x 106 of the various PMN for 2 h did not significantly reduce the log CFU (SFR3 CFU = 6.43, RB6 = 6.40, IAP+/+ = 6.48, and IAP-/- = 6.51, data not shown). These data confirm those of others that indicate that in vitro killing of S. aureus by PMN is not optimal unless Ab and other opsonins are present (29).
Exogenous administration of the CXC chemokine, MIP-2, decreases survival and increases the bacterial burden in mice infected with S. aureus
Because the above data indicated that limiting PMN migration into
the site of a S. aureus infection could be beneficial, we
asked whether promoting PMN migration would be deleterious. The murine
CXC chemokine, MIP-2, is a potent chemotactic factor for murine
neutrophils via the CXCR2 receptor (30). Therefore, we
asked whether exogenous addition of MIP-2 or another murine CXC
chemokine, KC, during a sublethal infection would be sufficient to
diminish host defense against this pathogen. We challenged wild-type
mice with a sublethal dose of DB and 4 h later injected i.p.
MIP-2, KC, or the CC chemokine MIP-1 as a control. Both MIP-2 and KC
significantly reduced survival of the mice as compared with the CC
chemokine control (Fig. 5A,
p = 0.0076). In addition, the timing of the MIP-2
administration was important. The maximal effect was observed at 4
h after the initiation of infection with minimal or no effect when
given at the time of infection. To prove that this effect was mediated
by PMN, we injected mice partially depleted of PMN by mAb RB6 vs SFR3
control with either saline or MIP-2 i.p. 4 h after challenge with
a sublethal dose of SA1 and assessed survival. Mice treated with
control mAb and saline all survived the infection, whereas mice treated
with control mAb and MIP-2 all succumbed to the infection by day 2
(Fig. 5B, p = 0.004). In contrast, 80% of
the mice that had been partially depleted of PMN with mAb RB6 before
treatment with MIP-2 survived this infection (p
= 0.024). Partial PMN depletion had no effect on the survival of
saline-injected mice. These data indicate that CXC chemokine
administration is sufficient to convert a sublethal S.
aureus infection into a lethal infection and that PMN are
primarily responsible for this deleterious effect.
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, MIP-2 significantly increased the
percentage and number of PMN present in the peritoneum in response to
infection with a sublethal challenge of both DB and 11D2. However, the
bacterial burden was significantly increased only with DB infection
(p = 0.006) and not with the sar-
mutant 11D2. Qualitatively identical data were obtained with SA1 and
with RN6390 infection as with DB (data not shown). Moreover, the number
of gentamicin-resistant, PMN-associated CFU in the presence of MIP-2
was greater for DB than for 11D2 (4.93 ± 0.42 log CFU vs
1.67 ± 0.11, n = 3). The number of
PMN-associated, gentamicin-resistant PMN was <1 log CFU for both
strains at this sublethal dose in the absence of exogenous MIP-2
administration (data not shown). In addition, MIP-2 administration
enhanced infection in IAP-/- mice (7.93 ±
0.44 log CFU vs saline control 5.37 ± 0.37 log CFU,
n = 3), indicating that the benefit of IAP deficiency
could be reversed by increased PMN migration (data not shown). These
data indicate that pathogenic strains of S. aureus
demonstrate enhanced survival in the presence of increased numbers of
PMN, both wild type and IAP deficient, and that the ability to do this
is regulated by sar.
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The above data indicated that CXC chemokine administration
increased the number of viable S. aureus inside PMN. This
raised the question of whether simply increasing the number of PMN was
sufficient or whether the chemokines played an additional role in
enhancing uptake of the bacteria. To address this, we developed an in
vitro assay with human PMN that would duplicate what we observed in
vivo in the mice. Human PMN were incubated with a 10:1 ratio of
S. aureus (strains DB, sar- 11D2, and SA1) in
the presence of the human CXC chemokine, IL-8, to mimic the relative
numbers of PMN, bacteria, and MIP-2 that we observed in the mice. After
4 h, the extracellular supernatant was separated from the PMN, and
both were subjected to gentamicin treatment. The extracellular CFU of
both DB and 11D2 were killed by gentamicin (Fig. 7). In contrast, the number of viable
CFUs associated with PMN after gentamicin treatment were significantly
higher for DB than for 11D2. Qualitatively identical data were obtained
with SA1 as with DB (data not shown). Minimal intracellular survival of
the bacteria was observed in buffer-treated, CC chemokine-stimulated,
or phorbol ester-stimulated PMN (data not shown). MIP-2 stimulation of
murine PMN also promoted uptake and intracellular survival of DB in
vitro (data not shown). To assess how blood-derived opsonins may affect
these results, we also included 10% fresh autologous human serum
containing agglutinating Ab and measured the number of
gentamicin-resistant, PMN-associated DB. In the presence of IL-8 alone,
the log CFU of the gentamicin-resistant, PMN-associated DB was
6.63 ± 0.21, n = 3. With the addition of 10%
serum the number was significantly reduced to 5.80 ± 0.014,
n = 3. However, the addition of serum-derived opsonins
did not ablate the ability of DB to remain viable inside PMN,
reflecting what we observed in vivo (Fig. 1A). These data
suggest that CXC chemokines both promote PMN migration and uptake of
S. aureus into a compartment that permits its survival.
Moreover, these data indicate that IL-8-stimulated human PMN, as well
as MIP-2-stimulated murine PMN, provide a sufficient environment for
the uptake and intracellular survival of S. aureus and that
intracellular survival is attenuated in a mutant sar-
strain.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Whereas S. aureus invasion of both epithelial cells and PMN
results in uptake into large vacuoles, our data with PMN differ from
those with epithelial cells in that an additional exogenous factor is
required. We found that incubation of PMN with the CXC chemokines,
IL-8, MIP-2, or KC, was required for optimal uptake and intracellular
survival. This is perhaps not surprising because chemotactic factors
promote membrane ruffling of PMN (31). In contrast,
neither the CC chemokine, MIP-1-a, which has minimal effects on PMN
function (30), nor phorbol esters, which stimulate many
PMN functions, were able to promote uptake and survival of S.
aureus. Because exogenous administration of CXC chemokines in vivo
enhanced the bacterial burden and the number of viable bacteria
associated with PMN, production of these chemokines in vivo may
contribute significantly to the pathogenesis of S. aureus
infection. Preliminary studies indicate that levels of MIP-2 and KC in
vivo are 1000-fold higher with wild-type S. aureus
infection than with the sar- mutant (H. D. Gresham,
unpublished observation). Therefore, sar may regulate
invasion and intracellular survival in PMN by affecting both the
synthesis of CXC chemokines and uptake by the PMN. In this regard,
recent evidence indicates that fibronectin binding protein, the
expression of which is regulated by sar (2, 3),
is required for efficient invasion of epithelial cells
(32). Studies are ongoing in our laboratories to assess
whether fibronectin-binding protein regulates both CXC chemokine
synthesis and intracellular survival in PMN in vivo.
Because we were interested in the consequences of the survival of S. aureus inside PMN as compared with other cell types, we asked whether limiting access to this privileged site would be beneficial to host defense. Although not particularly feasible with endothelial or epithelial cells, it is possible to test this by controlling the number of PMN at the site of infection. To achieve this, we used IAP-deficient mice and mice treated with low doses of mAb RB6 to limit the number of PMN at the site of infection. Both of these experimental approaches benefited host defense and reduced the bacterial burden. We believe that this enhanced host defense was provided by affecting PMN specifically because the benefit of both IAP deficiency and partial PMN depletion with mAb RB6 could be reversed by giving additional PMN and by promoting PMN migration with MIP-2 and KC administration. Although these experimental approaches reduced the number of viable bacteria, the precise mechanism for this remains unclear. The possibilities for this benefit include: 1) enhanced bactericidal killing by IAP-/- and RB6-treated PMN; 2) elimination of an intracellular reservoir of replicating organisms; 3) reduced CXC chemokine production and thus macropinocytic uptake; and 4) failure to sequester organisms away from the cells or agents that are competent for killing. We found that IAP-/- and RB6-treated PMN did not exhibit enhanced bactericidal activity in vitro. Moreover, we currently have no evidence to support replication of the organism inside PMN even though it may occur in other cells (17). Therefore, we believe that some combination of possibilities 3 and 4 are most likely to be operative. For example, reducing CXC chemokine synthesis which is essential for intracellular survival could facilitate opsonin-dependent uptake into tight phagosomes and thus result in destruction of the pathogen. In addition, reducing the number of PMN and thereby the number of intracellular organisms could simply increase the number of extracellular organisms accessible to extracellular bactericidal agents such as antimicrobial polypeptides (33) and type II phospholipase A2 (34). Further experimentation will be required to discriminate between these possibilities.
These experimental data raise the issue of whether survival of S. aureus inside PMN occurs during a natural infection. To begin to address this, we obtained fluid from an abscess that developed at the site of a surgical incision and that was shown subsequently to be infected with S. aureus. In this single very preliminary experiment, we found that PMN isolated from this site contained 5.5 x 106 CFU/1.0 x 107 PMN. Moreover, after incubation of the PMN with gentamicin, the CFU remained high at 4.3 x 106/ml (H. Gresham, unpublished observation). These data suggest that at sites of S. aureus-induced abscess formation, which occurs in the skin and in deep organs such as the liver, the appropriate environmental conditions may exist that would favor persistence of the organism by this mechanism. From our data, we hypothesize that this would minimally include: 1) expression by the bacteria of virulence determinants regulated by sar; 2) large numbers of viable PMN; 3) stimulated secretion of nanogram quantities of CXC chemokines; and 4) attenuated access of blood-derived opsonins to the site of infection. These conditions would favor uptake of the bacteria into macropinosomes as compared with tight phagosomes and thus perpetuate their survival even though PMN are present. Further experimentation will be required to determine whether this occurs during the course of natural infection at other sites.
Our data demonstrate that S. aureus has the ability to exploit the inflammatory response of the host to facilitate its own survival by gaining access to a locale within PMN where it is prevented from being killed. Moreover, the global regulator, sar, modulates this ability, indicating that it is a virulence determinant for this pathogen. In addition, our data suggest that successful therapeutic strategies for treating this infection could arise by manipulating the response of the host to block uptake of the pathogen into macropinosomes and/or to promote uptake into phagolysosomes for destruction.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hattie D. Gresham, Department of Molecular Genetics and Microbiology, Room 379, Basic Biomedical Science Building, 915 Camino de Salud, University of New Mexico, Albuquerque, NM 87131-5276. E-mail address:
3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophils; IAP, integrin-associate protein; HSA, human serum albumin; MIP, macrophage-inflammatory protein.
Received for publication October 25, 1999. Accepted for publication January 21, 2000.
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) is
compared with the number recovered (
). The number of PMN recovered
and the total CFU were significantly increased (p =
0.0044 and 0.0082, respectively). PMN containing viable intracellular
SA1 are sufficient to induce an influx of additional PMN and to
establish an environment that significantly increases the growth of the
pathogen.
) mice.
IAP-/- mice have significantly enhanced survival in
response to SA1 infection (p = 0.0055).
B, Comparison of the number of WBC per ml, number of PMN
per ml, percentage of PMN, and log CFU per ml in a 5-ml peritoneal
lavage from IAP+/+ (n = 5) (
, 
) or control SFR3 (
